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Ikuroh Ohsawa

Journal articles

Hiroko Hoshi, Wu Hao, Yoshinari Fujita, Atsushi Funayama, Yoshiteru Miyauchi, Kazuaki Hashimoto, Kana Miyamoto, Ryotaro Iwasaki, Yuiko Sato, Tami Kobayashi, Hiroya Miyamoto, Shigeyuki Yoshida, Tomoaki Mori, Hiroya Kanagawa, Eri Katsuyama, Atsuhiro Fujie, Kyoko Kitagawa, Keiichi I Nakayama, Toshihiro Kawamoto, Motoaki Sano, Keiichi Fukuda, Ikuroh Ohsawa, Shigeo Ohta, Hideo Morioka, Morio Matsumoto, Kazuhiro Chiba, Yoshiaki Toyama, Takeshi Miyamoto (2012)  Aldehyde-stress resulting from Aldh2 mutation promotes osteoporosis due to impaired osteoblastogenesis.   J Bone Miner Res Apr  
Abstract: Osteoporosis is a complex disease with various causes, such as estrogen loss, genetics and aging. Here we show that a dominant-negative form of aldehyde dehydrogenase 2 (ALDH2) protein, ALDH2*2, which is produced by a single nucleotide polymorphism (rs671), promotes osteoporosis due to impaired osteoblastogenesis. Aldh2 plays a role in alcohol-detoxification by acetaldehyde-detoxification, however, transgenic mice expressing Aldh2*2 (Aldh2*2 Tg) exhibited severe osteoporosis with increased levels of blood acetaldehyde without alcohol consumption, indicating that Aldh2 regulates physiological bone homeostasis. Wild-type osteoblast differentiation was severely inhibited by exogenous acetaldehyde, and osteoblastic markers such as osteocalcin, runx2 and osterix expression or phosphorylation of Smad1,5,8 induced by bone morphogenetic protein 2 (BMP2) was strongly altered by acetaldehyde. Acetaldehyde treatment also inhibits proliferation and induces apoptosis in osteoblasts. The Aldh2*2 transgene or acetaldehyde treatment induced accumulation of the lipid-oxidant 4-hydroxy-2-nonenal (4HNE) and expression of peroxisome proliferator-activated receptor gamma (PPARγ, a transcription factor that promotes adipogenesis and inhibits osteoblastogenesis. Anti-oxidant treatment inhibited acetaldehyde induced proliferation-loss, apoptosis and PPARγ expression and restored osteoblastogenesis inhibited by acetaldehyde. Treatment with a PPARγ inhibitor also restored acetaldehyde-mediated osteoblastogenesis inhibition. These results provide new insight into regulation of osteoporosis in a subset of individuals with ALDH2*2 and in alcoholic patients and suggest a novel strategy to promote bone formation in such osteopenic diseases. © 2012 American Society for Bone and Mineral Research.
Yasuhiro Terasaki, Ikuroh Ohsawa, Mika Terasaki, Mikiko Takahashi, Shinobu Kunugi, Kang Dedong, Hirokazu Urushiyama, Shunsuke Amenomori, Mayuko Kaneko-Togashi, Naomi Kuwahara, Arimi Ishikawa, Naomi Kamimura, Shigeo Ohta, Yuh Fukuda (2011)  Hydrogen therapy attenuates irradiation-induced lung damage by reducing oxidative stress.   Am J Physiol Lung Cell Mol Physiol 301: 4. L415-L426 Oct  
Abstract: Molecular hydrogen (H(2)) is an efficient antioxidant that diffuses rapidly across cell membranes, reduces reactive oxygen species (ROS), such as hydroxyl radicals and peroxynitrite, and suppresses oxidative stress-induced injury in several organs. ROS have been implicated in radiation-induced damage to lungs. Because prompt elimination of irradiation-induced ROS should protect lung tissue from damaging effects of irradiation, we investigated the possibility that H(2) could serve as a radioprotector in the lung. Cells of the human lung epithelial cell line A549 received 10 Gy irradiation with or without H(2) treatment via H(2)-rich PBS or medium. We studied the possible radioprotective effects of H(2) by analyzing ROS and cell damage. Also, C57BL/6J female mice received 15 Gy irradiation to the thorax. Treatment groups inhaled 3% H(2) gas and drank H(2)-enriched water. We evaluated acute and late-irradiation lung damage after H(2) treatment. H(2) reduced the amount of irradiation-induced ROS in A549 cells, as shown by electron spin resonance and fluorescent indicator signals. H(2) also reduced cell damage, measured as levels of oxidative stress and apoptotic markers, and improved cell viability. Within 1 wk after whole thorax irradiation, immunohistochemistry and immunoblotting showed that H(2) treatment reduced oxidative stress and apoptosis, measures of acute damage, in the lungs of mice. At 5 mo after irradiation, chest computed tomography, Ashcroft scores, and type III collagen deposition demonstrated that H(2) treatment reduced lung fibrosis (late damage). This study thus demonstrated that H(2) treatment is valuable for protection against irradiation lung damage with no known toxicity.
Tsuyoshi Mitsuishi, Kenji Kabashima, Hideaki Tanizaki, Ikuroh Ohsawa, Fumino Oda, Yuko Yamada, Yilinuer Halifu, Seiji Kawana, Toshihiko Kato, Kazumi Iida (2011)  Specific substance of Maruyama (SSM) suppresses immune responses in atopic dermatitis-like skin lesions in DS-Nh mice by modulating dendritic cell functions.   J Dermatol Sci 63: 3. 184-190 Sep  
Abstract: Specific substance of Maruyama (SSM) is a carcinostatic immunotherapeutic agent extracted from Mycobacterium tuberculosis. The efficacy of SSM induced interleukin(IL)-12 and IFN-γ production, and inhibition of IL-4, resulting in a shift from Th2 to Th1 in vivo.
Naomi Kamimura, Kiyomi Nishimaki, Ikuroh Ohsawa, Shigeo Ohta (2011)  Molecular hydrogen improves obesity and diabetes by inducing hepatic FGF21 and stimulating energy metabolism in db/db mice.   Obesity (Silver Spring) 19: 7. 1396-1403 Jul  
Abstract: Recent extensive studies have revealed that molecular hydrogen (H(2)) has great potential for improving oxidative stress-related diseases by inhaling H(2) gas, injecting saline with dissolved H(2), or drinking water with dissolved H(2) (H(2)-water); however, little is known about the dynamic movement of H(2) in a body. First, we show that hepatic glycogen accumulates H(2) after oral administration of H(2)-water, explaining why consumption of even a small amount of H(2) over a short span time efficiently improves various disease models. This finding was supported by an in vitro experiment in which glycogen solution maintained H(2). Next, we examined the benefit of ad libitum drinking H(2)-water to type 2 diabetes using db/db obesity model mice lacking the functional leptin receptor. Drinking H(2)-water reduced hepatic oxidative stress, and significantly alleviated fatty liver in db/db mice as well as high fat-diet-induced fatty liver in wild-type mice. Long-term drinking H(2)-water significantly controlled fat and body weights, despite no increase in consumption of diet and water. Moreover, drinking H(2)-water decreased levels of plasma glucose, insulin, and triglyceride, the effect of which on hyperglycemia was similar to diet restriction. To examine how drinking H(2)-water improves obesity and metabolic parameters at the molecular level, we examined gene-expression profiles, and found enhanced expression of a hepatic hormone, fibroblast growth factor 21 (FGF21), which functions to enhance fatty acid and glucose expenditure. Indeed, H(2) stimulated energy metabolism as measured by oxygen consumption. The present results suggest the potential benefit of H(2) in improving obesity, diabetes, and metabolic syndrome.
I-Chien Wu, Ikuroh Ohsawa, Noriyuki Fuku, Masashi Tanaka (2010)  Metabolic analysis of 13C-labeled pyruvate for noninvasive assessment of mitochondrial function.   Ann N Y Acad Sci 1201: 111-120 Jul  
Abstract: Mitochondria may play important roles in metabolic diseases, neurodegeneration, and aging. Therefore, noninvasive methods to assess mitochondrial metabolic function in humans are urgently needed. We demonstrated that the (13)C-breath test using [1-(13)C] acetate and [1-(13)C] pyruvate holds great potential for assessing the metabolic status in mitochondria. The (13)C-breath test allows us to assess dynamic metabolic pathways noninvasively within 60 min. This test will not only facilitate clinical evaluation of patients but also provide huge opportunities for further understanding of these conditions. The potential of the (13)C-breath test using (13)C-labeled pyruvate in humans cannot be overemphasized.
Sanae Nakajima, Ikuroh Ohsawa, Shigeo Ohta, Makoto Ohno, Toshio Mikami (2010)  Regular voluntary exercise cures stress-induced impairment of cognitive function and cell proliferation accompanied by increases in cerebral IGF-1 and GST activity in mice.   Behav Brain Res 211: 2. 178-184 Aug  
Abstract: Chronic stress impairs cognitive function and hippocampal neurogenesis. This impairment is attributed to increases in oxidative stress, which result in the accumulation of lipid peroxide. On the other hand, voluntary exercise enhances cognitive function, hippocampal neurogenesis, and antioxidant capacity in normal animals. However, the effects of voluntary exercise on cognitive function, neurogenesis, and antioxidants in stressed mice are unclear. This study was designed to investigate whether voluntary exercise cures stress-induced impairment of cognitive function accompanied by improvement of hippocampal neurogenesis and increases in antioxidant capacity. Stressed mice were exposed to chronic restraint stress (CRS), which consisted of 12h immobilization daily and feeding in a small cage, for 8 weeks. Exercised mice were allowed free access to a running wheel during their exposure to CRS. At the 6th week, cognitive function was examined using the Morris water maze (MWM) test. Daily voluntary exercise restored stress-induced impairment of cognitive function and the hippocampal cell proliferation of newborn cells but not cell survival. Voluntary exercise increased insulin-like growth factor 1 (IGF-1) protein and mRNA expression in the cerebral cortex and liver, respectively. In addition, CRS resulted in a significant increase in the number of 4-hydrosynonenal (4-HNE)-positive cells in the hippocampal dentate gyrus; whereas, voluntary exercise inhibited it and enhanced glutathione s-transferases (GST) activity in the brain. These findings suggest that voluntary exercise attenuated the stress-induced impairment of cognitive function accompanied by improvement of cell proliferation in the dentate gyrus. This exercise-induced improvement was attributed to exercise-induced enhancement of IGF-1 protein and GST activity in the brain.
Alexander M Wolf, Sadamitsu Asoh, Hidenori Hiranuma, Ikuroh Ohsawa, Kumiko Iio, Akira Satou, Masaharu Ishikura, Shigeo Ohta (2010)  Astaxanthin protects mitochondrial redox state and functional integrity against oxidative stress.   J Nutr Biochem 21: 5. 381-389 May  
Abstract: Mitochondria combine the production of energy with an efficient chain of reduction-oxidation (redox) reactions but also with the unavoidable production of reactive oxygen species. Oxidative stress leading to mitochondrial dysfunction is a critical factor in many diseases, such as cancer and neurodegenerative and lifestyle-related diseases. Effective antioxidants thus offer great therapeutic and preventive promise. Investigating the efficacy of antioxidants, we found that a carotenoid, astaxanthin (AX), decreased physiologically occurring oxidative stress and protected cultured cells against strong oxidative stress induced with a respiratory inhibitor. Moreover, AX improved maintenance of a high mitochondrial membrane potential and stimulated respiration. Investigating how AX stimulates and interacts with mitochondria, a redox-sensitive fluorescent protein (roGFP1) was stably expressed in the cytosol and mitochondrial matrix to measure the redox state in the respective compartments. AX at nanomolar concentrations was effective in maintaining mitochondria in a reduced state. Additionally, AX improved the ability of mitochondria to remain in a reduced state under oxidative challenge. Taken together, these results suggest that AX is effective in improving mitochondrial function through retaining mitochondria in the reduced state.
Tsuyoshi Mitsuishi, Yukie Iwabu, Kenzo Tokunaga, Tetsutaro Sata, Takehiko Kaneko, Kuniaki Ohara, Ikuroh Ohsawa, Fumino Oda, Yuko Yamada, Seiji Kawana, Kohji Ozaki, Mayuka Nakatake, Osamu Yamada (2010)  Combined analysis of cell growth and apoptosis-regulating proteins in HPVs associated anogenital tumors.   BMC Cancer 10: 03  
Abstract: The clinical course of human papillomavirus (HPV) associated with Bowenoid papulosis and condyloma acuminatum of anogenital tumors are still unknown. Here we evaluated molecules that are relevant to cellular proliferation and regulation of apoptosis in HPV associated anogenital tumors.
Hideaki Oharazawa, Tsutomu Igarashi, Takashi Yokota, Hiroaki Fujii, Hisaharu Suzuki, Mitsuru Machide, Hiroshi Takahashi, Shigeo Ohta, Ikuroh Ohsawa (2010)  Protection of the retina by rapid diffusion of hydrogen: administration of hydrogen-loaded eye drops in retinal ischemia-reperfusion injury.   Invest Ophthalmol Vis Sci 51: 1. 487-492 Jan  
Abstract: Retinal ischemia-reperfusion (I/R) injury by transient elevation of intraocular pressure (IOP) is known to induce neuronal damage through the generation of reactive oxygen species. Study results have indicated that molecular hydrogen (H(2)) is an efficient antioxidant gas that selectively reduces the hydroxyl radical (*OH) and suppresses oxidative stress-induced injury in several organs. This study was conducted to explore the neuroprotective effect of H(2)-loaded eye drops on retinal I/R injury.
Yuan Fu, Mikako Ito, Yasunori Fujita, Masafumi Ito, Masatoshi Ichihara, Akio Masuda, Yumi Suzuki, Satoshi Maesawa, Yasukazu Kajita, Masaaki Hirayama, Ikuroh Ohsawa, Shigeo Ohta, Kinji Ohno (2009)  Molecular hydrogen is protective against 6-hydroxydopamine-induced nigrostriatal degeneration in a rat model of Parkinson's disease.   Neurosci Lett 453: 2. 81-85 Apr  
Abstract: Molecular hydrogen serves as an antioxidant that reduces hydroxyl radicals, but not the other reactive oxygen and nitrogen species. In the past year, molecular hydrogen has been reported to prevent or ameliorate eight diseases in rodents and one in human associated with oxidative stress. In Parkinson's disease, mitochondrial dysfunction and the associated oxidative stress are major causes of dopaminergic cell loss in the substantia nigra. We examined effects of approximately 50%-saturated molecular hydrogen in drinking water before or after the stereotactic surgery on 6-hydroxydopamine-induced nigrostrital degeneration in a rat model of Parkinson's disease. Methamphetamine-induced behavioral analysis showed that molecular hydrogen prevented both the development and progression of the nigrostrital degeneration. Tyrosine hydroxylase staining of the substantia nigra and striatum also demonstrated that pre- and post-treatment with hydrogen prevented the dopaminergic cell loss. Our studies suggest that hydrogen water is likely able to retard the development and progression of Parkinson's disease.
Yutaka Harita, Hidetake Kurihara, Hidetaka Kosako, Tohru Tezuka, Takashi Sekine, Takashi Igarashi, Ikuroh Ohsawa, Shigeo Ohta, Seisuke Hattori (2009)  Phosphorylation of Nephrin Triggers Ca2+ Signaling by Recruitment and Activation of Phospholipase C-{gamma}1.   J Biol Chem 284: 13. 8951-8962 Mar  
Abstract: A specialized intercellular junction between podocytes, known as the slit diaphragm (SD), forms the essential structural frame-work for glomerular filtration in the kidney. In addition, mounting evidence demonstrates that the SD also plays a crucial role as a signaling platform in physiological and pathological states. Nephrin, the major component of the SD, is tyrosine-phosphorylated by a Src family tyrosine kinase, Fyn, in developing or injured podocytes, recruiting Nck to Nephrin via its Src homology 2 domain to regulate dynamic actin remodeling. Dysregulated Ca(2+) homeostasis has also been implicated in podocyte damage, but the mechanism of how podocytes respond to injury is largely unknown. Here we have identified phospholipase C-gamma1 (PLC-gamma1) as a novel phospho-Nephrin-binding protein. When HEK293T cells expressing a chimeric protein consisting of CD8 and Nephrin cytoplasmic domain (CD) were treated with anti-CD8 and anti-mouse antibodies, clustering of Nephrin and phosphorylation of Nephrin-CD were induced. Upon this clustering, PLC-gamma1 was bound to phosphorylated Nephrin Tyr-1204, which induced translocation of PLC-gamma1 from cytoplasm to the CD8/Nephrin cluster on the plasma membrane. The recruitment of PLC-gamma1 to Nephrin activated PLC-gamma1, as detected by phosphorylation of PLC-gamma1 Tyr-783 and increase in inositol 1,4,5-trisphosphate level. We also found that Nephrin Tyr-1204 phosphorylation triggers the Ca(2+) response in a PLC-gamma1-dependent fashion. Furthermore, PLC-gamma1 is significantly phosphorylated in injured podocytes in vivo. Given the profound effect of PLC-gamma in diverse cellular functions, regulation of the Ca(2+) signaling by Nephrin may be important in modulating the glomerular filtration barrier function.
Naomi Nakashima-Kamimura, Takashi Mori, Ikuroh Ohsawa, Sadamitsu Asoh, Shigeo Ohta (2009)  Molecular hydrogen alleviates nephrotoxicity induced by an anti-cancer drug cisplatin without compromising anti-tumor activity in mice.   Cancer Chemother Pharmacol 64: 4. 753-761 Sep  
Abstract: Cisplatin is a widely used anti-cancer drug in the treatment of a wide range of tumors; however, its application is limited by nephrotoxicity, which is affected by oxidative stress. We have reported that molecular hydrogen (H(2)) acts as an efficient antioxidant (Ohsawa et al. in Nat Med 13:688-694, 2007). Here we show that hydrogen efficiently mitigates the side effects of cisplatin by reducing oxidative stress.
Kazufumi Nagata, Naomi Nakashima-Kamimura, Toshio Mikami, Ikuroh Ohsawa, Shigeo Ohta (2009)  Consumption of molecular hydrogen prevents the stress-induced impairments in hippocampus-dependent learning tasks during chronic physical restraint in mice.   Neuropsychopharmacology 34: 2. 501-508 Jan  
Abstract: We have reported that hydrogen (H(2)) acts as an efficient antioxidant by gaseous rapid diffusion. When water saturated with hydrogen (hydrogen water) was placed into the stomach of a rat, hydrogen was detected at several microM level in blood. Because hydrogen gas is unsuitable for continuous consumption, we investigated using mice whether drinking hydrogen water ad libitum, instead of inhaling hydrogen gas, prevents cognitive impairment by reducing oxidative stress. Chronic physical restraint stress to mice enhanced levels of oxidative stress markers, malondialdehyde and 4-hydroxy-2-nonenal, in the brain, and impaired learning and memory, as judged by three different methods: passive avoidance learning, object recognition task, and the Morris water maze. Consumption of hydrogen water ad libitum throughout the whole period suppressed the increase in the oxidative stress markers and prevented cognitive impairment, as judged by all three methods, whereas hydrogen water did not improve cognitive ability when no stress was provided. Neural proliferation in the dentate gyrus of the hippocampus was suppressed by restraint stress, as observed by 5-bromo-2'-deoxyuridine incorporation and Ki-67 immunostaining, proliferation markers. The consumption of hydrogen water ameliorated the reduced proliferation although the mechanistic link between the hydrogen-dependent changes in neurogenesis and cognitive impairments remains unclear. Thus, continuous consumption of hydrogen water reduces oxidative stress in the brain, and prevents the stress-induced decline in learning and memory caused by chronic physical restraint. Hydrogen water may be applicable for preventive use in cognitive or other neuronal disorders.
Sanae Nakajima, Ikuroh Ohsawa, Kazufumi Nagata, Shigeo Ohta, Makoto Ohno, Tetsuo Ijichi, Toshio Mikami (2009)  Oral supplementation with melon superoxide dismutase extract promotes antioxidant defences in the brain and prevents stress-induced impairment of spatial memory.   Behav Brain Res 200: 1. 15-21 Jun  
Abstract: The purpose of this study was to investigate the effect of antioxidant ingestion on stress-induced impairment of cognitive memory. Male C57BL/6 mice were divided into four groups as follows: (1) control mice (C mice) fed in a normal cage without immobilization; (2) restraint-stressed (RS mice) fed in a small cage; (3) vitamin E mice (VE mice), mice were fed in a small cage with a diet supplemented with vitamin E; (4) GliSODin mice (GS mice) fed in a small cage with a diet supplemented with GliSODin. RS, VE and GS mice were exposed to 12 h of immobilization daily. Five weeks later, spatial learning was measured using the Morris Water Maze (MWM) test. After water maze testing, we performed immunohistochemical analysis using 4-hydroxy-2-noneral (4-HNE) and an anti-Ki67 antibody. 4-HNE is a marker of lipid peroxidation. RS mice showed impaired spatial learning performance and an increased number of 4-HNE-positive cells in the granule cell layer (GCL) of the hippocampal dentate gyrus when compared to C mice. Moreover, RS mice showed a decreased number of Ki67-positive cells in the subgranular zone (SGZ). GS mice showed better spatial learning memory than RS mice. The number of 4-HNE-positive cells in the GCL of GS mice was significantly less than that of RS mice. The number of Ki67-positive cells in the SGZ of GS mice was significantly greater than that of RS mice. These finding suggests that GliSODin prevents stress-induced impairment of cognitive function and maintains neurogenesis in the hippocampus through antioxidant activity.
Yoshihiko Suzuki, Motoaki Sano, Kentaro Hayashida, Ikuroh Ohsawa, Shigeo Ohta, Keiichi Fukuda (2009)  Are the effects of alpha-glucosidase inhibitors on cardiovascular events related to elevated levels of hydrogen gas in the gastrointestinal tract?   FEBS Lett 583: 13. 2157-2159 Jul  
Abstract: The major side-effect of treatment with alpha-glucosidase inhibitors, flatulence, occurs when undigested carbohydrates are fermented by colonic bacteria, resulting in gas formation. We propose that the cardiovascular benefits of alpha-glucosidase inhibitors are partly attributable to their ability to neutralise oxidative stress via increased production of H(2) in the gastrointestinal tract. Acarbose, which is an alpha-glucosidase inhibitor, markedly increased H(2) production, with a weaker effect on methane production. Our hypothesis is based on our recent discovery that H(2) acts as a unique antioxidant, and that when inhaled or taken orally as H(2)-dissolved water it ameliorates ischaemia-reperfusion injury and atherosclerosis development.
Yayoi Murakami, Ikuroh Ohsawa, Tadashi Kasahara, Shigeo Ohta (2009)  Cytoprotective role of mitochondrial amyloid beta peptide-binding alcohol dehydrogenase against a cytotoxic aldehyde.   Neurobiol Aging 30: 2. 325-329 Feb  
Abstract: Recent reports on amyloid beta peptide (A beta) binding-alcohol dehydrogenase (ABAD) have revealed the link of A beta with oxidative stress derived from mitochondria in the pathogenesis of Alzheimer's disease (AD). As a novel function of ABAD, we speculate that ABAD may detoxify aldehydes, such as 4-hydroxy-2-nonenal (4-HNE). To verify this speculation, we transfected cDNA encoding ABAD into cultured cells (HeLa and SH-SY5Y), where ABAD was localized to mitochondria. ABAD-transfectants decreased the levels of externally added 4-HNE in cultured medium as detected by TLC and became resistant against external 4-HNE. Moreover, ABAD suppressed the cytotoxic effects caused by cellular 4-HNE, which were produced through excess reactive oxygen species (ROS) by treatment with an inhibitor of mitochondrial respiration, antimycin A or by adding H(2)O(2). Catabolism of 4-HNE by ABAD was inhibited by A beta, resulting in the abolishment of the cytoprotective function by ABAD against ROS. These results propose an additional role of ABAD in neural cell death in AD: ABAD detoxifies aldehydes, such as 4-HNE derived from lipid peroxides in healthy brains, and inhibited by A beta in the development of AD.
Yuya Nakashima, Ikuroh Ohsawa, Fumiko Konishi, Takashi Hasegawa, Shoichiro Kumamoto, Yoshihiko Suzuki, Shigeo Ohta (2009)  Preventive effects of Chlorella on cognitive decline in age-dependent dementia model mice.   Neurosci Lett 464: 3. 193-198 Oct  
Abstract: Oxidative stress is one of the major causes of age-dependent memory loss and cognitive decline. Cytotoxic aldehydes are derived from lipid peroxides and their accumulation may be responsible for age-dependent neurodegeneration, including Alzheimer's disease. Since aldehyde dehydrogenases detoxify such aldehydes, we constructed transgenic mice with mitochondrial aldehyde dehydrogenase 2 (ALDH2) activity deficiency (DAL101 mice) as an age-dependent dementia model. This model animal is age-dependently progressed by persistent oxidative stress, and thus enables us to investigate foods that prevent dementia. Since Chlorella, a kind of alga, exhibits various anti-oxidative effects, we investigated whether Chlorella has the potential to prevent age-dependent cognitive impairment. We fed Chlorella to DAL101 mice and investigated its effects on oxidative stress and the progression of cognitive decline using the Morris water-maze and object recognition tests. The diet with Chlorella tended to reduce oxidative stress and significantly prevented the decline of cognitive ability, as shown by both methods. Moreover, consumption of Chlorella decreased the number of activated astrocytes in the DAL101 brain. These findings suggest that the prolonged consumption of Chlorella has the potential to prevent the progression of cognitive impairment.
Megumi Watanabe, Ken-ichiro Katsura, Ikuroh Ohsawa, Genki Mizukoshi, Kumiko Takahashi, Sadamitsu Asoh, Shigeo Ohta, Yasuo Katayama (2008)  Involvement of mitoKATP channel in protective mechanisms of cerebral ischemic tolerance.   Brain Res 1238: 199-207 Oct  
Abstract: Little work has been performed to determine roles of mitochondrial ATP-sensitive potassium channels (mitoK(ATP)) in ischemic preconditioning (IPC) in brain. To investigate the role on cerebral IPC, we examined effect of 5-hydroxydecanoate (5-HD), a selective mitoK(ATP) blocker, and diazoxide (DZX), a selective mitoK(ATP) opener on various IPC models. An IPC model with gerbil: 2 min bilateral common carotid arteries occlusion (BLCO)+24 h recovery+5 min BLCO. 5-HD, DZX, vehicle was administered 30 min before 5 min BLCO. Seven days later, surviving CA1 neurons were counted. A focal IPC model with rat: 15 min middle cerebral artery occlusion (MCAO)+48 h recovery+90 min MCAO. Twenty-four hours before 90 min MCAO, 5-HD, DZX, or vehicle was administered. One day after 90 min MCAO, neurological symptoms and infarct volumes were evaluated. An in vitro IPC model with primary neuronal cultures: 8 min oxygen-glucose deprivation (OGD)+24 h recovery+70 min OGD. Thirty minutes before 70 min OGD, 5-HD or DZX were added. One day later, surviving neurons were counted. Mitochondrial membrane potential was also monitored. 5-HD significantly attenuated the protective effect of IPC in gerbil model, rat model, and in vitro OGD model. DZX significantly facilitated the protective effect of IPC in gerbil and rat model. The mitochondrial membranes were depolarized with IPC, and 5-HD treatment significantly reduced this effect. These results strongly suggest that mitoK(ATP) channel activation plays a key role in development of a protective mechanism of cerebral IPC.
Kentaro Hayashida, Motoaki Sano, Ikuroh Ohsawa, Ken Shinmura, Kayoko Tamaki, Kensuke Kimura, Jin Endo, Takaharu Katayama, Akio Kawamura, Shun Kohsaka, Shinji Makino, Shigeo Ohta, Satoshi Ogawa, Keiichi Fukuda (2008)  Inhalation of hydrogen gas reduces infarct size in the rat model of myocardial ischemia-reperfusion injury.   Biochem Biophys Res Commun 373: 1. 30-35 Aug  
Abstract: Inhalation of hydrogen (H(2)) gas has been demonstrated to limit the infarct volume of brain and liver by reducing ischemia-reperfusion injury in rodents. When translated into clinical practice, this therapy must be most frequently applied in the treatment of patients with acute myocardial infarction, since angioplastic recanalization of infarct-related occluded coronary artery is routinely performed. Therefore, we investigate whether H(2) gas confers cardioprotection against ischemia-reperfusion injury in rats. In isolated perfused hearts, H(2) gas enhances the recovery of left ventricular function following anoxia-reoxygenation. Inhaled H(2) gas is rapidly transported and can reach 'at risk' ischemic myocardium before coronary blood flow of the occluded infarct-related artery is reestablished. Inhalation of H(2) gas at incombustible levels during ischemia and reperfusion reduces infarct size without altering hemodynamic parameters, thereby preventing deleterious left ventricular remodeling. Thus, inhalation of H(2) gas is promising strategy to alleviate ischemia-reperfusion injury coincident with recanalization of coronary artery.
Ken-ichiro Katsura, Kumiko Takahashi, Sadamitsu Asoh, Megumi Watanabe, Makoto Sakurazawa, Ikuroh Ohsawa, Takashi Mori, Hironaka Igarashi, Seiji Ohkubo, Yasuo Katayama, Shigeo Ohta (2008)  Combination therapy with transductive anti-death FNK protein and FK506 ameliorates brain damage with focal transient ischemia in rat.   J Neurochem 106: 1. 258-270 Jul  
Abstract: Many practical therapies have been explored as clinical applications for ischemic cerebral infarction; however, most are still insufficient to treat acute stroke. We show here a potential combination therapy in a rat focal ischemic model to improve neurological symptoms as well as to reduce infarct volumes at the maximum level. We applied protein transduction technology using artificial anti-death Bcl-xl derivative with three amino acid-substitutions (Y22F, Q26N and R165K) (FNK) protein fused with a protein-transduction-domain peptide (PTD-FNK). When PTD-FNK was administrated 1 h after initiating ischemia followed by the administration of an immunosuppressant FK506 with a 30-min time lag, infarct volumes of the total brain and cortex were markedly reduced to 27% and 14%, respectively. This procedure not only reduced the infarct volume and edema, but also markedly improved neurological symptoms. The therapeutic effect continued for at least 1 week after ischemia. FK506 inhibited the transduction of PTD-FNK in vitro, which explains the requirement of a time lag for the administration of FK506. An additional in vitro experiment showed that PTD-FNK, when administered 30 min before FK506, gave the maximal protective effect by reducing the intracellular calcium concentration. We propose that this combination therapy would provide a synergistic protective effect by both drugs, reducing adverse the effects of FK506.
Ikuroh Ohsawa, Kiyomi Nishimaki, Yayoi Murakami, Yuko Suzuki, Masahiro Ishikawa, Shigeo Ohta (2008)  Age-dependent neurodegeneration accompanying memory loss in transgenic mice defective in mitochondrial aldehyde dehydrogenase 2 activity.   J Neurosci 28: 24. 6239-6249 Jun  
Abstract: Oxidative stress may underlie age-dependent memory loss and cognitive decline. Toxic aldehydes, including 4-hydroxy-2-nonenal (HNE), an end product of lipid peroxides, are known to accumulate in the brain in neurodegenerative disease. We have previously shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) detoxifies HNE by oxidizing its aldehyde group. To investigate the role of such toxic aldehydes, we produced transgenic mice, which expressed a dominant-negative form of ALDH2 in the brain. The mice had decreased ability to detoxify HNE in their cortical neurons and accelerated accumulation of HNE in the brain. Consequently, their lifespan was shortened and age-dependent neurodegeneration and hyperphosphorylation of tau were observed. Object recognition and Morris water maze tests revealed that the onset of cognitive impairment correlated with the degeneration, which was further accelerated by APOE (apolipoprotein E) knock-out; therefore, the accumulation of toxic aldehydes is by itself critical in the progression of neurodegenerative disease, which could be suppressed by ALDH2.
Alexander M Wolf, Sadamitsu Asoh, Ikuroh Ohsawa, Shigeo Ohta (2008)  Imaging mitochondrial redox environment and oxidative stress using a redox-sensitive fluorescent protein.   J Nihon Med Sch 75: 2. 66-67 Apr  
Abstract: Redox-sensitive green fluorescent protein (roGFP) is a fluorescent protein in which two cysteines are placed adjacently in the barrel structure. Disulfide formation (oxidation) increases the absorption at short wavelengths (410 nm) at the expense of absorption at longer wavelengths (490 nm). The fluorescence ratio indicates reduction/oxidation, i.e., the redox potential at specific cellular locations.
Minoru Kogiku, Ikuroh Ohsawa, Koushi Matsumoto, Yuichi Sugisaki, Hiroshi Takahashi, Akira Teramoto, Shigeo Ohta (2008)  Prognosis of glioma patients by combined immunostaining for survivin, Ki-67 and epidermal growth factor receptor.   J Clin Neurosci 15: 11. 1198-1203 Nov  
Abstract: To confirm that survivin expression is a sensitive marker for the prognosis of glioma patients, surgically resected tissues of ninety-nine Japanese glioma patients using antibodies against survivin, Ki-67 and epidermal growth factor receptor (EGFR) were examined immunohistochemically. The median survival of patients with high survivin expression was significantly shorter than that with low expression (322 vs. 1084 days). Ki-67 expression was significantly associated with that of survivin, whereas no significant association between survivin and EGFR expressions was observed. Patients with low EGFR and low survivin expression survived longer than patients with high EGFR and low survivin expression (1509 vs. 795 days). These results indicate that survivin is a highly sensitive marker for glioma prognosis and suggest that the expression levels of survivin and other markers combined, including EGFR, might be a potent tool for the clinical prognosis of glioma patients.
Ikuroh Ohsawa, Kiyomi Nishimaki, Kumi Yamagata, Masahiro Ishikawa, Shigeo Ohta (2008)  Consumption of hydrogen water prevents atherosclerosis in apolipoprotein E knockout mice.   Biochem Biophys Res Commun 377: 4. 1195-1198 Dec  
Abstract: Oxidative stress is implicated in atherogenesis; however most clinical trials with dietary antioxidants failed to show marked success in preventing atherosclerotic diseases. We have found that hydrogen (dihydrogen; H(2)) acts as an effective antioxidant to reduce oxidative stress [I. Ohsawa, M. Ishikawa, K. Takahashi, M. Watanabe, K. Nishimaki, K. Yamagata, K. Katsura, Y. Katayama, S, Asoh, S. Ohta, Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals, Nat. Med. 13 (2007) 688-694]. Here, we investigated whether drinking H(2)-dissolved water at a saturated level (H(2)-water) ad libitum prevents arteriosclerosis using an apolipoprotein E knockout mouse (apoE(-/-)), a model of the spontaneous development of atherosclerosis. ApoE(-/-) mice drank H(2)-water ad libitum from 2 to 6 month old throughout the whole period. Atherosclerotic lesions were significantly reduced by ad libitum drinking of H(2)-water (p=0.0069) as judged by Oil-Red-O staining series of sections of aorta. The oxidative stress level of aorta was decreased. Accumulation of macrophages in atherosclerotic lesions was confirmed. Thus, consumption of H(2)-dissolved water has the potential to prevent arteriosclerosis.
Kei-ichi Fukuda, Sadamitsu Asoh, Masahiro Ishikawa, Yasuhiro Yamamoto, Ikuroh Ohsawa, Shigeo Ohta (2007)  Inhalation of hydrogen gas suppresses hepatic injury caused by ischemia/reperfusion through reducing oxidative stress.   Biochem Biophys Res Commun 361: 3. 670-674 Sep  
Abstract: We have recently showed that molecular hydrogen has great potential for selectively reducing cytotoxic reactive oxygen species, such as hydroxyl radicals, and that inhalation of hydrogen gas decreases cerebral infarction volume by reducing oxidative stress [I. Ohsawa, M. Ishikawa, K. Takahashi, M. Watanabe, K. Nishimaki, K. Yamagata, K.-I. Katsura, Y. Katayama, S. Asoh, S. Ohta, Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals, Nat. Med., 13 (2007) 688-694]. Here we show that the inhalation of hydrogen gas is applicable for hepatic injury caused by ischemia/reperfusion, using mice. The portal triad to the left lobe and the left middle lobe of the liver were completely occluded for 90min, followed by reperfusion for 180min. Inhalation of hydrogen gas (1-4%) during the last 190min suppressed hepatic cell death, and reduced levels of serum alanine aminotransferase and hepatic malondialdehyde. In contrast, helium gas showed no protective effect, suggesting that the protective effect by hydrogen gas is specific. Thus, we propose that inhalation of hydrogen gas is a widely applicable method to reduce oxidative stress.
Ikuroh Ohsawa, Masahiro Ishikawa, Kumiko Takahashi, Megumi Watanabe, Kiyomi Nishimaki, Kumi Yamagata, Ken-Ichiro Katsura, Yasuo Katayama, Sadamitsu Asoh, Shigeo Ohta (2007)  Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals.   Nat Med 13: 6. 688-694 Jun  
Abstract: Acute oxidative stress induced by ischemia-reperfusion or inflammation causes serious damage to tissues, and persistent oxidative stress is accepted as one of the causes of many common diseases including cancer. We show here that hydrogen (H(2)) has potential as an antioxidant in preventive and therapeutic applications. We induced acute oxidative stress in cultured cells by three independent methods. H(2) selectively reduced the hydroxyl radical, the most cytotoxic of reactive oxygen species (ROS), and effectively protected cells; however, H(2) did not react with other ROS, which possess physiological roles. We used an acute rat model in which oxidative stress damage was induced in the brain by focal ischemia and reperfusion. The inhalation of H(2) gas markedly suppressed brain injury by buffering the effects of oxidative stress. Thus H(2) can be used as an effective antioxidant therapy; owing to its ability to rapidly diffuse across membranes, it can reach and react with cytotoxic ROS and thus protect against oxidative damage.
Shigeo Ohta, Ikuroh Ohsawa (2006)  Dysfunction of mitochondria and oxidative stress in the pathogenesis of Alzheimer's disease: on defects in the cytochrome c oxidase complex and aldehyde detoxification.   J Alzheimers Dis 9: 2. 155-166 Jul  
Abstract: The mitochondrion is an organelle that plays a central role in energy production. It, at the same time, generates reactive oxygen species as by-products. Large-scale epidemiological case-control studies suggest the involvements of dihydrolipoamide succinyltransferase (DLST) of the mitochondrial Krebs cycle and mitochondrial aldehyde dehydrogenase-2 (ALDH2) in Alzheimer's disease (AD). The DLST gene has two gene-products, one of which, a novel gene product MIRTD, mediates the molecular assembly of the cytochrome c oxidase complex whose defect has been a candidate of the causes of AD. Since levels of MIRTD mRNA in the brains of AD patients were significantly low, a decrease in MIRTD could affect energy production. ALDH2, a matrix enzyme, was found to act as a protector against oxidative stress through oxidizing toxic aldehydes, such as 4-hydroxy-2-nonenal, that are spontaneously produced from lipid peroxides. Hence, a decrease in ALDH2 activity is proposed to contribute to AD. Indeed, transgenic mice with low activity of ALDH2 exhibited an age-dependent neurodegeneration accompanying memory loss. Since amyloid beta peptide has been recently shown to be present in neuronal mitochondria to decline energy production and enhance ROS production, it has become possible to link AD more closely with roles of mitochondria in the pathogenesis.
Yoshihiko Suzuki, Fujiko Ando, Ikuroh Ohsawa, Hiroshi Shimokata, Shigeo Ohta (2006)  Association of alcohol dehydrogenase 2*1 allele with liver damage and insulin concentration in the Japanese.   J Hum Genet 51: 1. 31-37 11  
Abstract: The Japanese have a polymorphism in the alcohol dehydrogenase 2 gene (ADH2). The alleles of ADH2 (ADH2*1 and ADH2*2) encode more active and less active forms for ethanol metabolism, respectively. We examined whether liver damage and the insulin-glucose axis vary according to ADH2 genotype in the Japanese. The 2,232 subjects (1,126 men and 1,106 women) were recruited from a population-based prospective cohort study. Clinical evaluations including alcohol consumption, percentage of alcohol drinkers, plasma glucose, HbA1c, insulin, AST, ALT, gamma-GTP, and prevalence of diabetes were compared among the ADH2 genotypes. The percentage of drinkers, alcohol consumption, AST, ALT, and gamma-GTP were higher in group ADH2*1/1 than in group ADH2*1/2 or ADH2*2/2 (all P < 0.05). Hence, ADH2*1/1 is associated with excess alcohol intake and liver disorders. However, the prevalence of diabetes did not differ among the three groups. For the glucose-insulin axis, we examined subjects who did not receive insulin therapy or oral anti-diabetes medication. While amounts of alcohol consumed and glucose levels were nearly the same between ADH*1/2 and ADH2*2/2, insulin concentrations were lower in ADH2*2/1 than in ADH2*2/2 (P < 0.05 in men). This finding suggests that the ADH2*1 allele is associated with a lower insulin concentration when alcohol intake is light or moderate. It also suggests that the genetic effect of ADH2*1 plays an important role in alcohol drinking behavior and in the occurrence of liver injury, but the effect is so mild that it does not influence the glucose-insulin axis or prevalence of diabetes.
Kentaro Sudo, Sadamitsu Asoh, Ikuroh Ohsawa, Daiya Ozaki, Kumi Yamagata, Hiromoto Ito, Shigeo Ohta (2005)  The anti-cell death FNK protein protects cells from death induced by freezing and thawing.   Biochem Biophys Res Commun 330: 3. 850-856 May  
Abstract: The FNK protein, constructed from anti-apoptotic Bcl-xL with enhanced activity, was fused with the protein transduction domain (PTD) of the HIV/Tat protein to mediate the delivery of FNK into cells. The fusion protein PTD-FNK was introduced into chondrocytes in isolated articular cartilage-bone sections, cultured neurons, and isolated bone marrow mononuclear cells to evaluate its ability to prevent cell death induced by freezing and thawing. PTD-FNK protected the cells from freeze-thaw damage in a concentration-dependent manner. Addition of PTD-FNK with conventional cryoprotectants (dimethyl sulfoxide and hydroxyethyl starch) increased surviving cell numbers around 2-fold compared with controls treated only with the cryoprotectants. Notably, PTD-FNK allowed CD34+ cells among bone marrow mononuclear cells to survive more efficiently (12-fold more than the control cells) from two successive freeze-thaw cycles. Thus, PTD-FNK prevented cell death induced by freezing and thawing, suggesting that it provides for the successful cryopreservation of biological materials.
Masaki Uematsu, Ikuroh Ohsawa, Toshiyuki Aokage, Kiyomi Nishimaki, Kouji Matsumoto, Hiroshi Takahashi, Sadamitsu Asoh, Akira Teramoto, Shigeo Ohta (2005)  Prognostic significance of the immunohistochemical index of survivin in glioma: a comparative study with the MIB-1 index.   J Neurooncol 72: 3. 231-238 May  
Abstract: Survivin has been identified as a protein expressed in cancer cells and a member of the inhibitor-of-apoptosis protein family. Recent studies suggest that the expression of survivin increases during the G2/M phase of the cell cycle, and may be used in clinical prognosis. We examined whether survivin expression in human gliomas would be a correlative of prognosis.
Toshiyuki Aokage, Ikuroh Ohsawa, Shigeo Ohta (2004)  Green fluorescent protein causes mitochondria to aggregate in the presence of the Bcl-2 family proteins.   Biochem Biophys Res Commun 314: 3. 711-716 Feb  
Abstract: Green fluorescent protein (GFP) has been widely used in a variety of experiments in cell biology. When cells were co-transfected with the GFP gene and the bcl-2 family genes bcl-2, bcl-x(L), and bax, mitochondria appeared to aggregate at the periphery of the nucleus specifically where GFP was expressed. Little aggregation was seen in the presence of other members of the GFP family, EGFP (enhanced GFP), ECFP (enhanced cyan variant), and EYFP (enhanced yellow-green variant). GFP but not EGFP seemed to promote cell death induced by pro-apoptotic Bax. Thus, GFP specifically promotes the aggregation of mitochondria when co-expressed with a member of the Bcl-2 family in association with apoptosis.
Ikuroh Ohsawa, Taiji Nishimura, Yukihiro Kondo, Go Kimura, Mitsuhiro Satoh, Ichiro Matsuzawa, Tsutomu Hamasaki, Shigeo Ohta (2004)  Detection of urine survivin in 40 patients with bladder cancer.   J Nihon Med Sch 71: 6. 379-383 Dec  
Abstract: We investigated whether urine survivin, an inhibitor of the apoptosis protein, is useful for diagnosing bladder tumor.
Y Suzuki, M Fujisawa, F Ando, N Niino, I Ohsawa, H Shimokata, S Ohta (2004)  Alcohol dehydrogenase 2 variant is associated with cerebral infarction and lacunae.   Neurology 63: 9. 1711-1713 Nov  
Abstract: The authors examined the association of the alcohol dehydrogenase 2 (ADH2) genotype with vascular events in community-dwelling Japanese (1,102 men/1,093 women). The allele ADH2*2 encodes an isozyme with a higher level of activity than ADH2*1. Here, the authors show that the ADH2*1 carriage is associated with high prevalence of cerebral infarction and lacunae in men. Multiple regression analyses confirmed that the risk of lacunae and cerebral infarction was increased by the ADH2*1 allele.
Shigeo Ohta, Ikuroh Ohsawa, Kouzin Kamino, Fujiko Ando, Hiroshi Shimokata (2004)  Mitochondrial ALDH2 deficiency as an oxidative stress.   Ann N Y Acad Sci 1011: 36-44 Apr  
Abstract: Mitochondrial aldehyde dehydrogenase 2 (ALDH2) plays a major role in ethanol metabolism. It is involved in acetaldehyde detoxification. A polymorphism of the ALDH2 gene is specific to North-East Asians. Sensitivity to ethanol is highly associated with this polymorphism (ALDH2(*)2 allele), which is responsible for a deficiency of ALDH2 activity. We first show that this deficiency influences the risk for late-onset Alzheimer's disease (LOAD) by a case-control study in a Japanese population. In a comparison of 447 patients with sex, age, and region-matched non-demented controls, the genotype frequency for the ALDH2(*)2 allele was significantly higher in the patients than in the controls (P=0.001). Next, we examined the combined effect of the ALDH2(*)2 and the apolipoprotein E4 allele (APOE-epsilon4), which has been confirmed to be a risk factor for LOAD. The ALDH2(*)2 allele more significantly affected frequency and age at onset in patients with APOE-epsilon4 than in those without it. These results indicate that the ALDH2 deficiency is a risk factor for LOAD, acting synergistically with the APOE-epsilon allele. Next, to elucidate the molecular mechanism involved, we obtained ALDH2-deficient cell lines by introducing mouse mutant ALDH2 cDNA into PC12 cells. We speculate that ALDH2 may act to oxidize toxic aldehyde derivatives. Then, we found that the ALDH2-deficient transfectants were highly vulnerable to exogenous 4-hydroxy-2-nonenal, an aldehyde derivative generated from peroxidized fatty acids. In addition, the ALDH2-deficient transfectants were sensitive to oxidative insult induced by antimycin A, accompanied by an accumulation of proteins modified with 4-hydroxy-2-nonenal. Mitochondrial ALDH2 functions as a protector against oxidative stress.
Kazutoshi Nakano, Ikuroh Ohsawa, Kumi Yamagata, Tomohiro Nakayama, Kaori Sasaki, Mikako Tarashima, Kayoko Saito, Makiko Osawa, Shigeo Ohta (2003)  Continuous culture of novel mitochondrial cells lacking nuclei.   Mitochondrion 3: 1. 21-27 Aug  
Abstract: We isolated stable cell lines, designated as mitochondrial cells, from cybrids obtained by fusing mitochondria-less HeLa cells with platelets from patients with Leigh syndrome, a subtype of mitochondrial encephalomyopathy. The cells contain a pathogenic point mutation, T9176C, in the mitochondrial DNA. Hematoxylin-eosin staining, confocal fluorescent microscopy and flow cytometry in fixed or living cells showed that the majority of these mitochondrial cells lack nuclear DNA and nuclei, but contain active mitochondria. Despite the absence of nuclear DNA, these cells can be continuously generated in culture. Therefore, it is likely that they arise from the minority of cells which possess a nucleus.
Ikuroh Ohsawa, Kiyomi Nishimaki, Chie Yasuda, Kouzin Kamino, Shigeo Ohta (2003)  Deficiency in a mitochondrial aldehyde dehydrogenase increases vulnerability to oxidative stress in PC12 cells.   J Neurochem 84: 5. 1110-1117 Mar  
Abstract: Mitochondrial aldehyde dehydrogenase 2 (ALDH2) plays a major role in acetaldehyde detoxification. The alcohol sensitivity is associated with a genetic deficiency of ALDH2. We have previously reported that this deficiency influences the risk for late-onset Alzheimer's disease. However, the biological effects of the deficiency on neuronal cells are poorly understood. Thus, we obtained ALDH2-deficient cell lines by introducing mouse mutant Aldh2 cDNA into PC12 cells. The mutant ALDH2 repressed mitochondrial ALDH activity in a dominant negative fashion, but not cytosolic activity. The resultant ALDH2-deficient transfectants were highly vulnerable to exogenous 4-hydroxy-2-nonenal, an aldehyde derivative generated by the reaction of superoxide with unsaturated fatty acid. In addition, the ALDH2-deficient transfectants were sensitive to oxidative insult induced by antimycin A, accompanied by an accumulation of proteins modified with 4-hydroxy-2-nonenal. Thus, these findings suggest that mitochondrial ALDH2 functions as a protector against oxidative stress.
Ikuroh Ohsawa, Kouzin Kamino, Keiko Nagasaka, Fujiko Ando, Naoakira Niino, Hiroshi Shimokata, Shigeo Ohta (2003)  Genetic deficiency of a mitochondrial aldehyde dehydrogenase increases serum lipid peroxides in community-dwelling females.   J Hum Genet 48: 8. 404-409 08  
Abstract: Mitochondrial aldehyde dehydrogenase 2 (ALDH2) plays a major role in acetaldehyde detoxification. The alcohol sensitivity is associated with a genetic deficiency of ALDH2. We and others have previously reported that such a deficiency influences the risk for late-onset Alzheimer's disease (LOAD), hypertension, and myocardial infarction. Then we tried to find phenotypes to which the ALDH2 polymorphism contributes by conducting several evaluations including biochemical and functional analyses of various tissues in a community-dwelling population. Several serum proteins, lipids, and lipid peroxides (LPO) levels showed differences between the nondefective (ALDH2*1/1) and defective (ALDH2*1/2 and ALDH2*2/2) ALDH2 individuals. However, alcohol-drinking behavior is known to affect these evaluations. Thus, we excluded the effects of alcohol-drinking behavior from the association with the ALDH2-deficient genotype through correction and found that the concentration of LPO was significantly lower in the nondefective ALDH2 females than the defective females. The effect of frequent alcohol-drinking behavior in males seems to override the phenotype of the high serum LPO level. These results indicate that the ALDH2 deficiency may enhance oxidative stress in vivo. Thus, these findings suggest that ALDH2 functions as a protector against oxidative stress and the decrease in protection may influence the onset of AD, hypertension, and myocardial infarction.
Sadamitsu Asoh, Ikuroh Ohsawa, Takashi Mori, Ken-Ichiro Katsura, Tomoharu Hiraide, Yasuo Katayama, Megumi Kimura, Daiya Ozaki, Kumi Yamagata, Shigeo Ohta (2002)  Protection against ischemic brain injury by protein therapeutics.   Proc Natl Acad Sci U S A 99: 26. 17107-17112 Dec  
Abstract: Preventing massive cell death is an important therapeutic strategy for various injuries and disorders. Protein therapeutics have the advantage of delivering proteins in a short period. We have engineered the antiapoptotic bcl-x gene to generate the super antiapoptotic factor, FNK, with a more powerful cytoprotective activity. In this study, we fused the protein transduction domain (PTD) of the HIVTat protein to FNK and used the construct in an animal model of ischemic brain injury. When added into culture media of human neuroblastoma cells and rat neocortical neurons, PTD-FNK rapidly transduced into cells and localized to mitochondria within 1 h. It protected the neuroblastomas and neurons against staurosporine-induced apoptosis and glutamate-induced excitotoxicity, respectively. The cytoprotective activity of PTD-FNK was found at concentrations as low as 0.3 pM. Additionally, PTD-FNK affected the cytosolic movement of calcium ions, which may relate to its neuroprotective action. Immunohistochemical analysis revealed that myc-tagged PTD-FNK (PTD-myc-FNK) injected i.p. into mice can have access into brain neurons. When injected i.p. into gerbils, PTD-FNK prevented delayed neuronal death in the hippocampus caused by transient global ischemia. These results suggest that PTD-FNK has a potential for clinical utility as a protein therapeutic strategy to prevent cell death in the brain.
I Ohsawa, C Takamura, S Kohsaka (2001)  Fibulin-1 binds the amino-terminal head of beta-amyloid precursor protein and modulates its physiological function.   J Neurochem 76: 5. 1411-1420 Mar  
Abstract: Genetic studies have implicated amyloid precursor protein (APP) in the pathogenesis of Alzheimer's disease. While accumulating lines of evidence indicate that APP has various functions in cells, little is known about the proteins that modulate its biological activity. Toward this end, we employed a two-hybrid system to identify potential interacting factors. We now report that fibulin-1, which contains repetitive Ca(2+)-binding EGF-like elements, binds to APP at its amino-terminal growth factor-like domain, the region that is responsible for its neurotrophic activities. Fibulin-1 expression in the brain is confined to neurons, and is not expressed significantly by astrocytes or microglia. Direct binding of fibulin-1 to the secreted form of APP (sAPP) was demonstrated with a pull-down assay using fragments of both fibulin-1 fused with glutathione-S transferase and sAPP, produced in bacteria and yeast, respectively. The fibulin-1/sAPP heteromer was shown to form in the conditioned medium of transfected COS-7 cells. Furthermore, fibulin-1 blocks sAPP-mediated proliferation of primary cultured rat neural stem cells. These results suggest that fibulin-1 may play a significant role in modulating the neurotrophic activities of APP.
I Ohsawa, C Takamura, T Morimoto, M Ishiguro, S Kohsaka (1999)  Amino-terminal region of secreted form of amyloid precursor protein stimulates proliferation of neural stem cells.   Eur J Neurosci 11: 6. 1907-1913 Jun  
Abstract: Beta-amyloid precursor protein (APP) has been reported to be expressed in the CNS from the early stages of development. However, the functional role of APP during early development remains unclear. In the present study, we found that the secreted form of APP (sAPP) significantly enhanced proliferation of neural stem cells. Cells were prepared from 13-day embryonic rat neocortex, which was dissected with a Pasteur pipette to make cell clusters. After 12 h of cultivation in the medium without serum, cells around the centre of the cluster were still nestin-positive proliferative cells, i.e. neural stem cells. To determine whether the proliferation of cells was regulated by sAPP, cultures were treated with recombinant sAPP695, the secreted form of human APP695 produced by yeast. Both DNA synthesis and expression of proliferating cell nuclear antigen markedly increased after 5 h of sAPP695 addition. The enhancement of DNA synthesis by sAPP695 stimulation was blocked by the 22C11 monoclonal antibody specific for the amino-terminal region of sAPP. Then, we examined the effect of the amino-terminal fragment of sAPP and the epitope peptide of 22C11 antibody, and found that both of them also promoted DNA synthesis, suggesting that the amino-terminal region of sAPP is responsible for the biological activity. Our findings indicate the possibility that sAPP enhances proliferation of neural stem cells in vivo and plays an important role during the early CNS development.
M Ishiguro, I Ohsawa, C Takamura, T Morimoto, S Kohsaka (1998)  Secreted form of beta-amyloid precursor protein activates protein kinase C and phospholipase Cgamma1 in cultured embryonic rat neocortical cells.   Brain Res Mol Brain Res 53: 1-2. 24-32 Jan  
Abstract: The secreted form of beta-amyloid precursor protein (sAPP) has been reported to exert various biological activities in cultured neurons. The signal transduction mechanisms underlying these physiological functions of sAPP remain unclear. We now report that treatment of neural cells with the secreted form of APP695 (sAPP695) leads to dose- and time-dependent increase in phosphorylation of the endogenous substrates with a molecular mass of 80, 57 and 43 kDa. Pretreatment of cells with protein kinase C (PKC) inhibitor H-7 reduced phosphorylation of the 80- and 43-kDa proteins in a dose-dependent manner. The effect of sAPP695 on the phosphorylation is mimicked by phorbol 12-myristate-13-acetate (PMA). Downregulation of PKC by prolonged treatment of cells with PMA abolished sAPP695-enhanced phosphorylation of the 80- and 43-kDa proteins, indicating PKC is involved in the sAPP695-enhanced phosphorylation of these proteins in the cells. We also suggest that the 80- and 43-kDa proteins phosphorylated by sAPP695-stimulation are the major PKC substrates myristoylated alanine-rich C-kinase substrate and growth-associated protein-43. Furthermore, we demonstrate that tyrosine phosphorylation of phospholipase Cgamma1 and formation of inositol 1,4,5-trisphosphate were increased by sAPP695-stimulation. These observations suggest that sAPP695 induces the activation of the signaling pathways through a stimulation of phosphoinositide-PKC cascade.
S Koizumi, M Ishiguro, I Ohsawa, T Morimoto, C Takamura, K Inoue, S Kohsaka (1998)  The effect of a secreted form of beta-amyloid-precursor protein on intracellular Ca2+ increase in rat cultured hippocampal neurones.   Br J Pharmacol 123: 8. 1483-1489 Apr  
Abstract: 1. The effects of secreted forms of beta-amyloid-precursor proteins (APP(S)s) on the intracellular Ca2+ concentration ([Ca2+]i) were investigated in rat cultured hippocampal neurones. APP695S, a secretory form of APP695, attenuated the increase in [Ca2+]i evoked by glutamate. In addition, APP695S itself evoked an increase in [Ca2+]i in 1 or 2 day-cultured hippocampal cells, but not in 7 to 13 day-cultured cells. 2. Eighty-one percent of neurones which were immunocytochemically positive for microtubule-associated protein 2 responded to APP695S with an increase in [Ca2+]i. 3. APP695S induced a transient rise in [Ca2+]i even in the absence of extracellular Ca2+ and produced an elevation in inositol-1,4,5-trisphosphate (IP3) in a concentration-dependent manner from 100 to 500 ng ml(-1). In the presence of extracellular Ca2+, APP695S caused a transient rise in [Ca2+]i followed by a sustained phase at high [Ca2+]i, suggesting Ca2+ entry from the extracellular space. 4. The [Ca2+]i elevation was mimicked by amino terminal peptides of APPs, but not by carboxy terminal peptides. 5. These results taken together suggest that APP695S induces an increase in [Ca2+]i in hippocampal neurones through an IP3-dependent mechanism that changes according to the stage of development.
T Morimoto, I Ohsawa, C Takamura, M Ishiguro, Y Nakamura, S Kohsaka (1998)  Novel domain-specific actions of amyloid precursor protein on developing synapses.   J Neurosci 18: 22. 9386-9393 Nov  
Abstract: The effect of the secretory form of amyloid precursor protein (sAPP) on synaptic transmission was examined by using developing neuromuscular synapses in Xenopus cell cultures. The frequency of spontaneous postsynaptic currents (SSCs) was reduced by the addition of sAPP, whereas the amplitude of impulse-evoked postsynaptic currents (ESCs) was increased by sAPP. These opposing effects on spontaneous versus evoked release were separated by using the specific domain of APP. The C-terminal fragment of sAPP (CAPP) only reduced SSC frequency and did not affect ESCs. By contrast, the N-terminal fragment of sAPP (NAPP) did not affect SSC frequency but did increase ESC amplitude. The reduction of SSC frequency by sAPP appears to be mediated by activation of potassium channels through a cGMP-dependent pathway, whereas the increase of ESC amplitude is mediated by a different pathway involving activation of protein kinase(s). These results suggest the potential role of sAPP as a modulator of synaptic activity by two specific domains.
T Morimoto, I Ohsawa, C Takamura, M Ishiguro, S Kohsaka (1998)  Involvement of amyloid precursor protein in functional synapse formation in cultured hippocampal neurons.   J Neurosci Res 51: 2. 185-195 Jan  
Abstract: Amyloid precursor protein (APP) is known to be widely expressed in neuronal cells, and enriched in the central and peripheral synaptic sites. Although it has been proposed that APP functions in synaptogenesis, no direct evidence has yet been reported. In this study we investigated the involvement of APP in functional synapse formation by monitoring spontaneous oscillations of intracellular Ca2+ concentration ([Ca2+]i) in cultured hippocampal neurons. As more and more neurons form synapses with each other during the culture period, increasing numbers of neuronal cells show synchronized spontaneous oscillations of [Ca2+]i. The number of neurons that showed synchronized spontaneous oscillations of [Ca2+]i was significantly lower when cultured in the presence of monoclonal antibody 22C11 against the N-terminal portion of APP. Moreover, incubation with excess amounts of the secretory form of APP or the N-terminal fragment of APP also inhibited the increase in number of neurons with synchronized spontaneous oscillations of [Ca2+]i. The addition of monoclonal antibody 22C11 or secretory form of APP did not, however, affect MAP-2-positive neurite outgrowth. These findings suggest that APP play a role in functional synapse formation during CNS development.
I Ohsawa, C Takamura, S Kohsaka (1997)  The amino-terminal region of amyloid precursor protein is responsible for neurite outgrowth in rat neocortical explant culture.   Biochem Biophys Res Commun 236: 1. 59-65 Jul  
Abstract: We have previously shown that secreted forms of beta-amyloid precursor protein (APP(s)s) promote neurite outgrowth in embryonic rat neocortical explant culture. To determine the region of APP(s) responsible for its biological activity, we produced both amino- and carboxyl-terminal regions of APP(s) using a yeast expression system. The purified fragment corresponding to the amino-terminal region (NAPP) enhanced neurite outgrowth of neocortical explants, but the carboxyl-terminal region fragment did not. The neurite-promoting activity of full length APP(s) and NAPP was blocked by the antibody, 22C11, specific for the amino-terminal region, and the 16-mer peptide of epitope for 22C11 also enhanced neurite outgrowth. However, the 17-mer peptide which contains RERMS sequence did not enhance the neurite outgrowth, but promoted the survival of neocortical neurons in dissociated culture. These findings suggested that the amino-terminal region is responsible for the neurite-promoting activity of APP(s)s.
M Shinoda, K Toide, I Ohsawa, S Kohsaka (1997)  Specific inhibitor for prolyl endopeptidase suppresses the generation of amyloid beta protein in NG108-15 cells.   Biochem Biophys Res Commun 235: 3. 641-645 Jun  
Abstract: A potent and specific prolyl endopeptidase inhibitor, JTP-4819, was used to investigate the role of prolyl endopeptidase in the generation of amyloid beta protein (A beta) from APP by NG108-15 cells. Synthetic substrates, 7-(succinyl-Ile-Ala)-4-methylcoumarinamide and Z(Val-Lys-Met)-4-methylcoumarinamide, respectively, corresponding to the C-terminal and N-terminal portions of A beta, were cleaved by NG108-15 cell lysates. Cleavage of the C-terminal portion, but not the N-terminal, was inhibited by JTP-4819 (IC50 = 0.6 nM). Western blot analysis showed that the A beta level in the culture medium of NG108-15 cells was increased by serum deprivation. JTP-4819 caused concentration (>10(-9) M)- and time-dependent inhibition of this serum deprivation-induced increase of A beta without having any effect on the level of the secretory form of APP. Using both specific anti-A beta (1-40) and anti-A beta (1-42) antisera, the A beta that increased with serum deprivation was confirmed to be A beta (1-40), suggesting that it might be produced by conversion of A beta (1-42) to A beta (1-40). These findings indicate that prolyl endopeptidase may be a key enzyme in the production of A beta by NG108-15 cells and that A beta secretion can be modulated by a prolyl endopeptidase inhibitor.
I Ohsawa, Y Hirose, M Ishiguro, Y Imai, S Ishiura, S Kohsaka (1995)  Expression, purification, and neurotrophic activity of amyloid precursor protein-secreted forms produced by yeast.   Biochem Biophys Res Commun 213: 1. 52-58 Aug  
Abstract: The secreted form of amyloid precursor protein (APPs) including most of the extracellular domain of APP is released from the cell surface, suggesting physiological significance of APPs in vivo. We used the methylotrophic yeast Pichia pastoris as a host system for the production of recombinant APPs (rAPPs). Two rAPPss derived from isoforms of APP (APP695 and APP770) were secreted into the culture medium from the yeast, which carried cDNA encoding the N-terminal portion of APP under the control of a P. pastoris alcohol oxidase promoter. Like APPss produced by the transfected COS-1 cells, the purified rAPPss from yeast were shown to be biologically active in terms of neurite outgrowth of embryonic rat neocortical explants. These rAPPss could be valuable tools for investigating the biological functions of APPss.
D Speck, I Ohsawa, R Gloeckler, M Zinsius, S Bernard, C Ledoux, T Kisou, K Kamogawa, Y Lemoine (1991)  Isolation of Bacillus sphaericus biotin synthesis control mutants: evidence for transcriptional regulation of bio genes.   Gene 108: 1. 39-45 Dec  
Abstract: The genes involved in biotin synthesis have recently been isolated from Bacillus sphaericus [Gloeckler et al., Gene 87 (1990) 63-70]. Sequence analysis revealed that they are organized into two gene clusters, designated bioXWF and bioDAYB. The 5'-noncoding region of the bioD locus fused to the xylE reporter gene was inserted into the Gram-positive pUB110 replicon and the resulting plasmid was introduced into B. sphaericus IFO3525. Transformants expressed the xylE gene only if the biotin concentration in the growth medium remained below 50 ng/ml. After mutagenesis, colonies were screened for their ability to express the chromogenic marker in the presence of an excess of biotin. Most of these mutants escaped biotin repression of xylE gene expression. Classical genetic analysis showed they formed two main categories: chromosomal mutations, pleiotropically acting in trans on both bioXWF and bioDAYB 5'-noncoding regions, in which a 15-bp region common to both promoters represented a hot-spot for the second class of plasmid-associated mutations. These results, completed by the identification of transcription start points for the bioD and bioX genes, strongly suggest that this 15-bp sequence overlaps the site of a biotin-mediated negative regulation circuit controlling the transcription of the bio genes.
R Gloeckler, I Ohsawa, D Speck, C Ledoux, S Bernard, M Zinsius, D Villeval, T Kisou, K Kamogawa, Y Lemoine (1990)  Cloning and characterization of the Bacillus sphaericus genes controlling the bioconversion of pimelate into dethiobiotin.   Gene 87: 1. 63-70 Mar  
Abstract: Using 8.8 kb of genetic information from Bacillus sphaericus, it was possible to confer to Escherichia coli bio- strains, including delta bioA-D, bioC-, bioH-, the ability to convert exogenous pimelate into biotin. The bio genes were borne on two recombinant plasmids with inserts of 4.3 kb and 4.5 kb, which had been isolated from a genomic bank of HindIII-digested B. sphaericus DNA, by phenotypic complementation of various E. coli bio mutants. The B. sphaericus bioD and bioA genes were unambiguously identified within the 4.3-kb insert and shown to be closely linked to bioY (coding for a protein with a presently unknown function) and to bioB [Ohsawa et al., Gene 80 (1989) 39-48]. These genes are clustered in the order bioDAYB. The 4.5-kb fragment contains genetic information for three different proteins, the products of bioX, bioW and bioF. Complementation studies using an E. coli bioF mutant and a B. subtilis bio112TG3 strain, revealed that the third ORF of this cluster encodes 7-keto-8-aminopelargonic acid synthetase. A combination of bioW and bioF allows an efficient complementation of E. coli bioC and bioH mutants, provided that pimelate is added to the biotin-depleted growth medium. No function could be identified for the product of bioX. The gene order of this cluster is bioXWF. By sequence analysis, the two cloned DNA fragments were shown to bear overlapping open reading frames and secondary structures at their 3' ends, typical of transcription terminators.(ABSTRACT TRUNCATED AT 250 WORDS)
I Ohsawa, D Speck, T Kisou, K Hayakawa, M Zinsius, R Gloeckler, Y Lemoine, K Kamogawa (1989)  Cloning of the biotin synthetase gene from Bacillus sphaericus and expression in Escherichia coli and Bacilli.   Gene 80: 1. 39-48 Aug  
Abstract: Biotin synthetase (BS) catalyses the biotransformation of dethiobiotin (DTB) to biotin. Here we report the cloning, characterization and expression of the gene encoding BS of Bacillus sphaericus. A recombinant plasmid pSB01, containing an 8.2-kb DNA fragment from B. sphaericus, was isolated by phenotypic complementation of an Escherichia coli bioB strain. Nucleotide sequence analysis of this fragment and N-terminal sequence determination of the recombinant protein product revealed that the bioB gene of B. sphaericus consists of a 996-bp open reading frame which is closely associated with at least one other gene. E. coli cells transformed with a bioB expression vector performed efficient bioconversion of DTB to biotin under defined culture conditions. Biotin production from transformed Bacillus subtilis and B. sphaericus recombinant strains was also demonstrated. Comparison of the amino acid sequences of BS from E. coli and B. sphaericus revealed extensive similarity.
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